Part of the work performed during development of this invention utilized U.S. Government funds. The U.S. Government has certain rights in this invention.
1. Field of the Invention
The present invention relates to novel parathyroid hormone-related peptide (PTHrP) analogs. In particular, the invention relates to PTHrP analogs having one or more amino acid substitutions that confer PTH-2 receptor antagonist or agonist properties to the analog.
2. Description of Related Art
Parathyroid hormone (PTH) is a major regulator of calcium homeostasis whose principal target cells occur in bone and kidney. Regulation of calcium concentration is necessary for the normal function of the gastrointestinal, skeletal, neurologic, neuromuscular, and cardiovascular systems. PTH synthesis and release are controlled principally by the serum calcium level; a low level stimulates and a high level suppresses both hormone synthesis and release. PTH, in turn, maintains the serum calcium level by directly or indirectly promoting calcium entry into the blood at three sites of calcium exchange: gut, bone, and kidney. PTH contributes to net gastrointestinal absorption of calcium by favoring the renal synthesis of the active form of vitamin D. PTH promotes calcium resorption from bone by inhibiting osteoblasts and, indirectly, by stimulating differentiation of the bone-resorbing cells, osteoclasts. It also mediates at least three main effects on the kidney: stimulation of tubular calcium reabsorption, enhancement of phosphate clearance, and promotion of an increase in the enzyme that completes synthesis of the active form of vitamin D. PTH exerts these effects primarily through receptor-mediated activation of adenylate cyclase, although receptor-mediated activation of phospholipase C by PTH has also been reported (Hruska et al., J Clin. Invest. 79:230 (1987)).
Disruption of calcium homeostasis may produce many clinical disorders (e.g., severe bone disease, anemia, renal impairment, ulcers, myopathy, and neuropathy) and usually results from conditions that produce an alteration in the level of parathyroid hormone. Hypercalcemia is a condition that is characterized by an elevation in the serum calcium level. It is often associated with primary hyperparathyroidism in which an excess of PTH production occurs as a result of a lesion (e.g., adenoma, hyperplasia, or carcinoma) of the parathyroid glands. Another type of hypercalcemia, humoral hypercalcemia of malignancy (HHM) is the most common paraneoplastic syndrome. It appears to result in most instances from the production by tumors (e.g., squamous, renal, ovarian, or bladder carcinomas) of a novel class of protein hormone which shares amino acid homology with PTH. These PTH-related proteins (PTHrP) appear to mimic certain of the renal and skeletal actions of PTH and are believed to interact with the PTH receptor in these tissues. PTHrP is normally found at low levels in many tissues, including keratinocytes, brain, pituitary, parathyroid, adrenal cortex, medulla, fetal liver, osteoblast-like cells, and lactating mammary tissues. In many HHM malignancies, PTHrP is found in the circulatory system at high levels, thereby producing the elevated calcium levels associated with HHM.
The pharmacological profiles of PTH and PTHrP are nearly identical in most in vitro assay systems, and elevated blood levels of PTH (i.e., primary hyperparathyroidism) or PTHrP (i.e., HHM) have comparable effects on mineral ion homeostasis (Broadus, A. E. and Stewart, A. F., xe2x80x9cParathyroid hormone-related protein: Structure, processing and physiological actions,xe2x80x9d in Basic and Clinical Concepts, Bilzikian, J. P. et al., eds., Raven Press, N.Y. (1994), pp. 259-294; Kronenberg, H. M. et al., xe2x80x9cParathyroid hormone: Biosynthesis, secretion, chemistry and action,xe2x80x9d in Handbook of Experimental Pharmacology, Mundy, G. R. and Martin, T. J., eds., Springer-Verlag, Heidelberg (1993), pp.185-201). The similarities in the biological activities of the two ligands can be explained by their interaction with a common receptor, the PTH/PTHrP receptor, which is expressed abundantly in bone and kidney (Urena, P. et al., Endocrinology 134:451-456 (1994)).
The binding of either radiolabeled PTH-(1-34) or PTHrP-(1-36) to the PTH/PTHrP receptor is competitively inhibited by either unlabeled ligand (Jxc3xcppner, H. et al., J. Biol. Chem. 263:8557-8560 (1988); Nissenson, R. A. et al., J. Biol. Chem. 263:12866-12871 (1988)). Thus, the recognition sites for the two ligands in the PTH/PTHrP receptor probably overlap. In both PTH and PTHrP, the 15-34 region contains the principal determinants for binding to the PTH/PTHrP receptor. Although these regions show only minimal sequence homology (only 3 amino acid identities), each 15-34 peptide can block the binding of either PTH-(1-34) or PTHrP-(1-34) to the PTH/PTHrP receptor (Nussbaum, S. R. et al., J. Biol. Chem. 255:10183-10187 (1980); Caulfield, M. P. et al., Endocrinology 127:83-87 (1990); Abou-Samra, A.-B. et al., Endocrinology 125:2215-2217 (1989)). Further, the amino terminal portion of each ligand is required for bioactivity, and these probably interact with the PTH/PTHrP receptor in similar ways, since 8 of 13 of these residues are identical in PTH and PTHrP.
The PTH/PTHrP receptor is a member of a distinct family of G protein-coupled receptors (Jxc3xcppner, H. et al., Science 254:1024-1026 (1991); Abou-Samra, A. B. et al., Proc. Natl. Acad. Sci (USA) 89:2732-2736 (1992)) that includes receptors for other peptide hormones such as secretin (Ishihara, T. et al., EMBO J. 10:1635-1641 (1991)), calcitonin (Lin, H. Y. et al., Science 254:1022-1024 (1991)) and glucagon (Jelinek, L. J. et al., Science 259:1614-1616 (1993)). Using degenerate oligonucleotides corresponding to conserved regions of the PTH/secretin/calcitonin receptor family, Usdin et al. has identified a new receptor cDNA derived from rat and human brain that was most closely related to the PTH/PTHrP receptor (51% overall amino acid sequence identity) (Usdin, T. et al., J Biol. Chem. 270:15455-15458 (1995)). This new receptor, the PTH-2 receptor, responded efficiently, potently, and specifically to PTH-(1-34), but interestingly, did not respond at all to PTHrP. Id. This observation implied that structural differences in the PTH and PTHrP ligands determined selectivity for interaction with the PTH-2 receptor. The PTH-2 receptor was found predominantly in the brain and pancreas. Id.
Since the PTH-2 receptor was found in brain and pancreas, it is likely to have a physiological role in the normal functioning of those organs. Diseases associated with brain and pancreatic dysfunction may in fact be explained by the altered action of the PTH-2 receptor. Such diseases could then be treated with a PTH-2 receptor selective antagonist. Other antagonists that are available are not selective and thus, are not desired because they would also antagonize the PTH/PTHrP receptor, which could have negative consequences in terms of calcium regulation and skeletal function.
Accordingly, there is a need in the art for the development of PTH-2 receptor selective agonists and antagonists: (1) to assist in further elucidating the biological role of the PTH-2 receptor; (2) to map specific sites of ligand-receptor interaction; and (3) as potential new therapeutic compositions that can be used in the treatment of disorders associated with altered action or genetic mutation of the PTH-2 receptor.
To begin to define residues involved in the ligand-specificity of the PTH-2 receptor, the inventors studied the interaction of several PTH/PTHrP hybrid ligands and other related peptide analogs, with the human PTH-2 receptor. The results showed that two sites in PTH and PTHrP fully account for the different potencies that the two ligands exhibited with PTH-2 receptors; residue 5 (His in PTHrP and Ile in PTH) determined signaling capability, while residue 23 (Phe in PTHrP and Trp in PTH) determined binding affinity. By changing these two residues of PTHrP (i.e., residue 5 and 23) to the corresponding residues of PTH, the inventors were able to convert PTHrP into a ligand that avidly bound to the PTH-2 receptor, and fully and potently stimulated cAMP formation. Changing residue 23 alone yielded [Trp23]hPTHrP-(1-36), which was an antagonist for the PTH-2 receptor, but a fill agonist for the PTH/PTHrP receptor. Accordingly, the inventors concluded that residues 5 and 23 in PTH and PTHrP play key roles in signaling and binding interactions, respectively, with the PTH-2 receptor.
Thus, in accordance with one aspect of the present invention, there is provided a novel PTHrP analog that is a potent PTH-2 receptor agonist, as well as a potent PTH/PTHrP receptor agonist. In a preferred embodiment, the PTHrP analog is altered at residues 5 and 23 to the corresponding residues of PTH. Most preferably, the invention includes PTHrP analogs having an amino acid substitution of histidine for isoleucine at position 5 of PTHrP, as well as phenylalanine for tryptophan at position 23 of PTHrP. In a particular embodiment, the PTHrP analog is [Ile5, Trp23]PTHrP-(1-36).
In accordance with another aspect of the present invention, there is provided a novel PTHrP analog that is a potent PTH-2 receptor selective antagonist. In a preferred embodiment, the analog is altered at PTHrP residue 23 to the corresponding residue of PTH. Most preferably, the invention includes PTHrP analogs having a single amino acid substitution of phenylalanine for tryptophan at position 23 of PTHrP. In a particular embodiment, the PTHrP analog is [Trp23]PTHrP-(1-36).
In accordance with yet a further aspect of the invention, there is provided a method for treating a medical disorder that results from altered or excessive action of the PTH-2 receptor, comprising administering to a patient a therapeutically efficient amount of a PTHrP analog, sufficient to inhibit activation of the PTH-2 receptor of said patient. In a preferred embodiment, the PTHrP analog used in the method has a single amino acid substitution of phenylalanine for tryptophan at position 23 of PTHrP. In a particular embodiment, the PTHrP analog is [Trp23]PTHrP-(1-36).
In accordance with yet a further aspect of the invention, there is provided a method for treating osteoporosis, comprising administering to a patient a therapeutically efficient amount of a PTHrP analog, sufficient to activate the PTH/PTHrP receptor and PTH-2 receptor of said patient. In a preferred embodiment, the PTHrP analog used in the method has an amino acid substitution of histidine for isoleucine at position 5 of PTHrP, as well as phenylalanine for tryptophan at position 23 of PTHrP. In a particular embodiment, the PTHrP analog is [Ile5, Trp23]PTHrP-(1-36).
In addition, any other amino-acid substitutions at positions 5 and/or 23, which do not destroy the ability of the PTHrP analog to antagonize or agonize the PTH-2 receptor (as determined by assays known to the skilled artisan and discussed below), are included within the scope of the present invention.